The study of proteins in living cells and in tissues (proteomics) is an active area of clinical and basic scientific research because metabolic control m cells and tissues is exercised at the protein level. For example, comparison of the levels of protein expression between healthy and diseased tissues, or between pathogenic and nonpathogenic microbial strains, can speed the discovery and development of new drug compounds or agricultural products. Further, analysis of the protein expression pattern in diseased tissues or in tissues excised from organisms undergoing treatment can also serve as diagnostics of disease states or the efficacy of treatment strategies, as well as provide prognostic information regarding suitable treatment modalities and therapeutic options for individual patients. Still further, identification of sets of proteins in samples derived from microorganisms (e.g., bacteria) can provide a means to identify the species and/or strain of microorganism as well as, with regard to bacteria, identify possible drug resistance properties of such species or strains.
Because it can used to provide detailed protein and peptide structural information, mass spectrometry (MS) is currently considered to be a valuable analytical tool for biochemical mixture analysis and protein identification. Conventional methods of protein analysts therefore often combine two-dimensional (2D) gel electrophoresis, for separation and quantification, with mass spectrometric identification of proteins. Also, capillary liquid chromatography as well as various other “front-end” separation or chemical fractionation techniques have been combined with electrospray ionization tandem mass spectrometry for large-scale protein identification without gel electrophoresis. Using mass spectrometry, qualitative differences between mass spectra can be identified, and proteins corresponding to peaks occurring in only some of the spectra serve as candidate biological markers.
The term “top-down proteomics” refers to methods of analysis in which protein samples are introduced intact into a mass spectrometer, without prior enzymatic, chemical or other means of digestion. Top-down analysis enables the study of the intact proteins, allowing identification, primary structure determination and localization of post-translational modifications (PTMs) directly at the protein level. Top-down proteomic analysis typically consists of introducing an intact protein into the ionization source of a mass spectrometer, determining the intact mass of the protein, fragmenting the protein ions and measuring the mass-to-charge ratios (m/z) and abundances of the various fragments so-generated This sequence of instrumental steps is commonly referred to as tandem mass spectrometry or, alternatively, “MS/MS” analysis. Such techniques may be advantageously employed for polypeptide studies. The resulting fragmentation is many times more complex than the fragmentation of simple peptides. The interpretation of such fragment mass spectra generally includes comparing the observed fragmentation pattern to either a protein sequence database that includes compiled experimental fragmentation results generated from known samples or, alternatively, to theoretically predicted fragmentation patterns. For example, Liu et al. (“Top-Down Protein Identification/Characterization of a Priori Unknown Proteins via Ion Trap Collision-Induced Dissociation and Ion/Ion Reactions in a Quadrupole/Time-of-Flight Tandem Mass Spectrometer”, Anal. Chem. 2009, 81, 1433-1441) have described top-down protein identification and characterization of both modified and unmodified unknown proteins with masses up to ≈28 kDa.
An advantage of a top-down analysis over a bottom-up analysis is that a protein may be identified directly, rather than inferred as is the case with peptides in a so-called “bottom-up” analysis. Another advantage is that alternative forms of a protein, e.g. post-translational modifications and splice variants, may be identified. However, top-down analysis has a disadvantage when compared to a bottom-up analysis in that many proteins can be difficult to isolate and purify. Thus, each protein in an incompletely separated mixture can yield, upon mass spectrometric analysis, multiple ion species, each species corresponding to a different respective degree of protonation and a different respective charge state, and each such ion species can give rise to multiple isotopic variants. A single MS spectrum measured in a top-down analysis can easily contain hundreds to even thousands of peaks which belong to different analytes—all interwoven over a given m/z range in which the ion signals of very different intensities overlap.
Front-end sample fractionation, such as two-dimensional gel electrophoresis or liquid chromatography, when performed prior to MS analysis, can reduce the complexity of various individual mass spectra. Nonetheless, the mass spectra of such sample fractions may still comprise the signatures of multiple proteins and/or polypeptides. The general technique of conducting mass spectrometry (MS) analysis of ions generated from compounds separated by liquid chromatography (LC) may be referred to as “LC-MS”. If the mass spectrometry analysis is conducted as tandem mass spectrometry (MS/MS), then the above-described procedure may be referred to as “LC-MS/MS”. In conventional LC-MS/MS experiments a sample is initially analyzed by mass spectrometry to determine mass-to-charge ratios (m/z) of ions derived from a sample and to identify (i.e., select) mass spectral peaks of interest. The sample is then analyzed further by product ion MS/MS scans on the selected peak(s). More specifically, in a first stage of analysis, frequently referred to as “MS1”, a full-scan mass spectrum, comprising an initial survey scan, is obtained. This full-scan spectrum is then followed by the selection of one or more precursor ion species. The precursor ions of the selected species are subjected to fragmentation such as may be accomplished employing a collision cell or employing another form of fragmentation cell such as surface-induced dissociation, electron-transfer dissociation or photo-dissociation. In a second stage, the resulting fragment (product) ions are detected for further analysis (frequently referred to as either “MS/MS” or “MS2”) using either the same or a second mass analyzer. A resulting product spectrum exhibits a set of fragmentation peaks (a fragment set) which, in many instances, may be used as a means to derive structural information relating to the precursor ion species.
FIG. 1A illustrates a hypothetical experimental situation in which different fractions, attributable to different analyte species, are chromatographically well resolved (in time) upon introduction into a mass spectrometer. Curves A10 and A12 represent a hypothetical concentration of each respective analyte at various times, where concentration is indicated as a percentage on a relative intensity (R.I.) scale and time is plotted along the abscissa as retention time. The curves A10 and A12 may be readily determined from measurements of total ion current input into a mass spectrometer. A threshold intercity level A8 of the total ion element is set below which only MS1 data is acquired. As a first analyte—detected as peak A10—elutes, the total ion current intensity crosses the threshold A8 at time t1. When this occurs, an on-board processor or other controller of the mass spectrometer may initiate one or more MS/MS spectra to be acquired. Subsequently, the leading edge of another elution peak A12 is detected. When the total ion current once again breaches the threshold intensity A8 at time t3, one or more additional MS/MS scans are initiated. Generally, the peaks A10 and A12 will correspond to the elution of different analytes and, thus, different precursor ions are selected for fragmentation during the elution of the first analyte (between time t1 and time t2) than are selected during the elution of the second analyte (between time t3 and time t4). Because the different precursor ions will, in general, comprise different m/z ratios and different charge states, the experimental conditions inquired to produce optimum fragmentation may differ between the two different elution periods.
In a more-complex mixture of analytes, there may be components whose elution peaks completely overlap, as illustrated in the graph of ion current intensity versus retention time in FIG. 1B. In this example elution peak A11 represents the ion current attributable to a precursor ion generated from a first analyte and the elution peak A13 represents the ion current attributable to a different precursor ion generated from a second analyte, where the masses and/or charge states of these different precursor ions are different from one another. In the hypothetical situation shown in FIG. 1B, there is almost perfect overlap of the elution of the compounds that give rise to the different ions, with the mass spectral intensity of the first precursor ion always being greater than that of the second precursor ion during the course of the co-elution. At any time daring the co-elution of the two analytes—for example, between time t6 and time t7—a mass spectrum of all precursor ions may appear as is hypothetical shown in FIG. 1C, with the set of lines indicated by envelope 78 arising from ionization of the first analyte and the set of lines indicated by envelope 76 arising from ionization of the second analyte. Under these conditions, automated mass spectral analysis must be able to not only distinguish between different precursor ions associated with the different respective analytes but must also be able to adjust the collision energy that is imparted to the different precursor ions during mass spectral analysis such that each ion is optimally fragmented. Indeed, as noted below, proper scaling of applied collision energy is important even when analytes are not co-eluting. The correct scaling is of particular importance, regardless of relative elution timing, when the characteristics of multiple analytes (e.g., MW and/or z) are significantly different.
One common method of causing ion fragmentation in MS-MS analyses is collision induced dissociation (CID), in which a population of analyte precursor ions are accelerated into target neutral gas molecules such as nitrogen (N2) or argon (Ar), thereby imparting internal vibrational energy to precursor ions which can lead to bond breakage and dissociation. The fragment ions are analyzed so as to provide useful information regarding the structure of the precursor ion. The term “collision induced dissociation” includes techniques in which energy is imparted to precursor ions by means of a resonance excitation process, which may be referred to as RE-CID techniques. Such resonant-excitation methods include application of an auxiliary alternating current voltage (AC) to trapping electrodes in addition to a main RF trapping voltage. This auxiliary voltage typically has relatively low amplitude (on the order of 1 Volt (V)) and duration on the order of tens of milliseconds. The frequency of this auxiliary voltage is chosen to match an ion's frequency of motion, which in turn is determined by the main trapping field amplitude, frequency and the ion's mass-to-charge ratio (m/z). As a consequence of the ion's motion being in resonance with the applied voltage, the ion's energy increases, and its amplitude of motion grows.
FIG. 2 schematically illustrates another method of collision induced dissociation, which is sometimes referred to as higher-energy collisional dissociation (HCD). In the HCD method selected ions are either temporarily stored in or caused to pass through a multipole ion storage device 52, which may, for instance, comprise a multipole ion trap. At a certain time, an electrical potential on a gate electrode assembly 54 is changed so as to accelerate the selected precursor ions 6 out of the ion storage device and into a collision cell 56 containing molecules 8 of an inert target gas. The ions are accelerated so as to collide with the target molecules at a kinetic energy that is determined by the difference in the potential offsets between the collision cell and the storage device.
It is highly desirable, when using either HCD or RE-CID to generate fragment ions in MS/MS experiments, to set instrumentation so as to impart a correct amount of collision energy to selected precursor ions. For HCD, the collision energy (CE) is set by setting the potential difference through which ions are accelerated into the HCD cell. There they collide one or more times with the resident gas until they exceed a vibrational energy threshold for bond cleavage to produce dissociation product ions. Product ions may retain enough kinetic energy that further collisions result in serial dissociation events. The optimal collision energy varies according to the properties of the selected precursor ions. Setting the HCD collision energy too high can result in such serial dissociation events, producing an abundance of small, non-specific product ion species. Conversely, setting this potential too low will result in a paucity of informative product ions ail together since the mass spectral signature of at least some fragment ions may be weak or absent. In either case, one would not be able to gain sufficient structural information about the precursor ion from the product ion spectrum to provide for identification or structural or sequence) elucidation. Analytes of different size, structure, and charge capacity dissociate to a different degree at any given CE. Therefore, using just a single collision energy setting for all precursor ions dining the course of an automated mass spectral analysis experiment presents the risk that the degree of fragmentation will be sub-optimal or non-acceptable for some ions. Nonetheless, mass spectral analysis programs are often performed on samples or sample fractions having a reduced chemical diversity for a variety of reasons (e.g., ionization, chromatography, fragmentation, etc). Reducing the chemical diversity increases the likelihood of setting an appropriate collision energy through tuning collision energy on similar analytes.
Although resonant excitation CID (RE-CID) and HCD produce similar mass spectra from the same charge from the same protein, the exact collision energy optimum needed to produce the maximum amount of structural information can vary greatly. In the case of RE-CID, since the applied auxiliary frequency is at the same fundamental frequency as the motion of a precursor ion, the internal energy of the precursor ion is increased to point that a minimum energy of dissociation is reached and product ions are produced. As the applied energy is increased the degree of fragmentation reaches a maximum and plateaus as the precursor ion is depleted. If the applied fragmentation energy is further increased there is typically no change in the relative abundances of the various product ions. Instead, the relative abundances of product ions remain approximately constant as fragmentation energy is increased beyond the onset of the plateau region and little to no additional relevant structural information is obtained front this process.
In contrast, in the case of HCD fragmentation, the collisional activation process is a function only of the electrical potential difference between the HCD cell and an adjacent ion optical element. Therefore, any product ions formed in the HCD cell can undergo further fragmentation depending on their excess internal energy. Since the HCD process involves the use of nitrogen as a collision gas versus that of helium typically used in RE-CID experiments, higher energies and more structural information can be gained from the HCD process, provided that a near-optimal collision energy is applied. In the RE-CID process, increase of applied collision energyy beyond its optimal value decreases the amount of remaining precursor ion but does not significantly change the relative amounts of fragment ions. In HCD fragmentation, increase of applied collision energy beyond its optimal value often causes further fragmentation of fragment ions.
FIG. 3A shows a general comparison between the effect of increasing energy on the number of identifiable protein fragment ions generated by HCD fragmentation (curve 151) and the effect of increasing energy on the number of such identifiable ions generated by RE-CID fragmentation (curve 152). Curve illustrates the effect of changing applied resonance energy on the fragmentation of a precursor ion derived from the protein myoglobin. In this example, when the collision energy is increased beyond 25% RCE, the amount of structural information remains relatively constant. In contrast, when the HCD process is employed (curve 151), there is a sharply defined maximum in structural information content obtained for an HCD energy of approximately 28% RCE. At collision energies either less than or exceeding this optimal RCE setting, there can be a dramatic decrease in the quality of structural information obtained from an HCD experiment.
The effect of changing applied HCD fragmentation energy is well illustrated in the fragmentation of the +8 charge state precursor ion from the protein ubiquitin, as illustrated in the product ion mass spectra of FIGS. 3B-3D. FIG. 3B shows a limited number of fragment ions produced from fragmentation of this ion using a sub-optimal RCE setting of 25%. In many experimental situations, such limited fragmentation will not allow for the proper identification of the protein from either searching a standard tandem mass spectrometry library or using sequence information front available databases. However, when the RCE setting is changed to 30%, the HCD fragmentation of the same precursor ion is optimal and the resulting product ion mass spectrum (FIG. 3C) exhibits a rich array of fragments of various charge states that enable the protein to be identified using any one of several approaches. Finally, as shown in FIG. 3D, a further increase of the RCE setting to 40% causes an over-fragmentation situation in which the majority of the generated product ions are singly charged low mass fragments that are more indicative of the amino acid composition of the protein than the actual protein sequence itself. Therefore it is highly desirable that collision energies for the HCD fragmentation of unknown proteins and complex mixtures be adjusted in real time so as to maximize the information content available.
U.S. Pat. No. 6,124,591, in the name of inventors Schwartz et al., describes a method of generating product ions by RE-CID in a quadrupole ion trap, in which the amplitude of the applied resonance excitation voltage is substantially linearly related to precursor-ion m/z ratio. The techniques described in U.S. Pat. No. 6,124,591 attempt to normalize out the primary variations in optimal resonance excitation voltage amplitude for differing ions, and also the variations due to instrumental differences. Schwartz et al. further found that the effects of the contributions of varying structures, charge states and stability on the determination of applied collision energy are secondary in nature and that these secondary effects may be modeled by simple correction factors.
According to the teaching of Schwartz et al., the substantially linear relationship between optimal applied CE and m/z is simply and rapidly calibrated on a per instrument basis. The accompanying FIG. 4A schematically illustrates the principles of generation and use of the calibration curve. Initially, a calibration curve for a particular mass spectral instrument is generated by fitting a linear relationship to calibration data in which a particular percentage of reduction (such as 90% reduction) of precursor-ion intensity is observed. This linear relationship is illustrated as line 22 in FIG. 4A. Schwartz et al. found that a two-point calibration is sufficient to characterize the linear relationship and that, more simply, a one-point calibration may be used if an intercept for the line is fixed at a certain value or at zero. In a typical calibration, the intercept of the calibration line 22 is assumed to be at the origin, as shown in FIG. 4A, and a one-point calibration includes determination or calculation of the applied collision energy at a reference point 29 at a specified reference mass-to-charge ratio (m/z)0. Typically, the reference point is at m/z=500 Da and the reference collision energy value measured at or extrapolated to 500 Da during calibration may be denoted as CE500.
Once an instrumental calibration has been determined, subsequent operation of the mass spectrometer does not generally employ the full CE values suggested by the line 22 but, instead, employs a relative collision energy (RCE) value, expressed as a percentage of the CE value of the value given by line 22 at any given m/z. For example, lines 24, 26 and 28 shown in FIG. 4A represent RCE values of 75%, 50% and 25%, respectively. Subsequently, a user may simply specify a desired value of RCE. The secondary effects of precursor-ion charge state, z, on optimal applied CE are accounted for by simple scalar charge correction factors, f(z). These general relationships, initially determined for RE-CID fragmentation base been also found to be valid for HCD fragmentation. With these simplifications, the absolute collision energy, CEactual, which is expressed in electron volts for HCD fragmentation, that is applied to each precursor is then automatically set according to the following equation:
                              CE          actual                =                  RCE          ×                      CE            500                    ×                      [                                          (                                  m                  z                                )                            /              500                        ]                    ×                      f            ⁡                          (              z              )                                                          Eq        .                                  ⁢        1            where CEactual is the appled collision energy, generally expressed in electron-Volts (eV), RCE is Relative Collision Energy, a percentage value that is generally user-defined for each experiment and f(z) is a charge correction factor. Table 1 in FIG. 4B lists the accepted charge correction factors. Note that both the numerator and denominator of the fraction in brackets are expressed in units of Daltons, Da (or, more accurately, thomsons, Th). Although this equation is typically sufficient to fine tune the absolute CE applied to samples within a narrow range of precursor ion characteristics, it should be noted that, as f(z) yields a fixed value for z≥5, the collision energies are usually too high for heavier molecules with higher charge states (such as proteins and polypeptides), leading to an over-fragmentation of those species.
Recently, mass spectral analysis of intact proteins and polypeptides has gained significant popularity. For such applications, analytes within a sample can range dramatically in size, structure, and charge capacity, and therefore require very different collision energies to achieve the same extent of dissociation. It has been found that the equation above does not sufficiently normalize collision energy for all precursors in samples of polypeptides or intact proteins, even if the range of charge factors is extended and extrapolated for charge states above +5. Therefore, a revised model is required for these particular analytes.